Antibodies are powerful tools for analyzing mutations in antigens (Pollock et al. 1987) and they have been successfully used for selecting antigenic variants, also known as escape mutants, of influenza virus (Gerhard and Webster 1978, Lubeck et al. 1980, Yewdel et al. 1986), rabies virus (Wiktor and Koprowski 1980) and measles virus (Birrer et al. 1981). NDV escape mutants were produced by Russel (1983), Abenes et al. (1986), Meulemans et al. (1987) and Yussof et al. (1989). Meulemans et al. (1987) showed that NDV escape mutants may be more, or less pathogenic than the parental virus strains (Meulemans et al. 1987).
The production of antibody resistant virus strains to deliberately attenuate the parental virus and make it suitable as in ovo vaccine was never suggested. Benejean et al. (EP0583998) attenuated the rabies virus by producing an escape mutant and used it as a vaccine. They never suggested or implied the use of this technique to produce escape mutants of avian viruses possibly useful for in ovo vaccination.
In ovo vaccination technology using approved vaccine is a safe, efficacious, and convenient method for vaccination of poultry (Ricks et al. 1999, U.S. Pat. No. 6,032,612, AO1K45/00C). In 1999, more than 80% of the U.S. broiler industry had converted to the in ovo vaccination process to control Marek's disease (Ricks et al. 1999).
Studies within the last few years have shown that only few live vaccines that are routinely administered to hatched chickens may also be injected into embryonated eggs during late stages of embryonation without a toxic effect. The turkey herpes virus (HVT, Sharma and Burmester, 1982), and infectious bursal disease virus (IBDV) strains of low virulence (Sharma, 1985) can be used as embryo vaccines to induce active protection against the homologous strains.
IBDV strains of moderate virulence such as 2512 (Sharma 1985), commercial infectious bronchitis virus (IBV) strains such as Massachusetts 41 (Wakenell and Sharma 1986) and Newcastle disease virus strains such as the B1 (Ahmad and Sharma, 1992, 1993) and the La Sota strain, cannot be employed for in ovo vaccination in their current form due to embryonic toxicity. Attenuation of the virus strains currently used for post hatch vaccination is thus required to obtain strains with reduced pathogenicity to the avian embryo.
Wakenell and Sharma (1986) reduced the pathogenicity to the embryo of the Massachusetts 41 IBV strain using a tissue culture attenuation system. At the 40th passage in chicken kidney tissue culture, the virus became apathogenic for the embryos and embryonic vaccination induced IBV specific antibody production and protection against virulent Massachusetts 41 IBV at 4 weeks of age.
Treatment of the B1 strain of NDV with the alkylating agent ethylmethane sulfonate markedly reduced the virulence of this strain for the 18-day chick embryo, and in ovo vaccination with this strain resulted in NDV specific antibody production and protection against challenge with the Texas GB strain (Ahmad and Sharma 1992). Further, it was claimed (EP0848956 A1) that a vaccine preparation containing Newcastle disease viruses of the strain NDW was particularly suited for in ovo application.
Finally, Mebatsion and Schrier (EP1074614A1) produced a NDV La Sota mutant, which is suited as vaccine candidate for in ovo vaccination. The mutant expresses reduced levels of V protein and can safely be administered to chicken embryos before hatch. No antibody-based selection was used to obtain these strains.
The formation of a complex between a measured amount of antibody with IBDV neutralizing activity and a specific amount of IBDV neutralized the pathogenicity of the IBDV and made it useful as in ovo vaccine (U.S. Pat. No. 5,871,748, Whithfill et al. 1992, 1995, Haddad et al. 1997).
In order to reduce the economic losses due to Newcastle disease in the commercial poultry industry, chickens currently have to be vaccinated against the Newcastle disease virus. It may be advantageous to use embryo vaccination for said purpose, in particular since in ovo injection can be done using semiautomatic machines with multiple injection heads allowing individual vaccination.
However, many vaccines used conventionally for post-hatch vaccination of birds cannot be used for in ovo vaccination. It is therefore an aim of the present invention to provide attenuated Newcastle disease virus strains which can be effectively used as a vaccine in avian species, administrable post hatch or in ovo. Another aim of the present invention is to provide a general method for selecting attenuated avian virus strains, and in particular attenuated strains of Newcastle disease virus.